(M1030-07-46) Leveraging Carnitine-Functionalized Lipid Nanocarrier-Based Targeted Delivery of A1874 PROTAC for Glioblastoma

Purpose: Glioblastoma(GBM) is the most aggressive and lethal form of primary brain cancer, characterized by rapid progression, therapeutic resistance, and a median survival of only 12–15 months despite maximal standard-of-care interventions. One of the most formidable challenges in GBM treatment is the limited ability of therapeutic agents to penetrate the blood-brain barrier (BBB), reinforced by the tumor’s inherent resistance mechanisms. Notably, resistance to frontline temozolomide(TMZ) therapy remains a major clinical challenge, markedly diminishing therapeutic efficacy and contributing to consistently poor patient outcomes. These limitations highlight a critical need for novel therapeutic strategies that can bypass physiological barriers and address the molecular complexity of GBM. Recent innovations in targeted protein degradation have introduced proteolysis-targeting chimeras (PROTACs) as transformative agents in oncology. Among them, A1874, a heterobifunctional molecule, has demonstrated potent, selective degradation of oncogenic drivers such as BRD4 and MDM2, both of which are implicated in GBM proliferation, survival, and therapeutic resistance. However, A1874 falls under the “beyond Rule of 5” (bRo5) chemical space, presenting significant challenges in terms of aqueous solubility, membrane permeability, and CNS delivery. To address these obstacles , we propose development of targeted self-nanoemulsifying drug delivery system (SNEDDS)—termed PROnano—specifically engineered for brain-targeted delivery of A1874. This study aimed to enhance the brain-targeted delivery of A1874 by leveraging a rationally designed nanocarrier system functionalized with Palmitoyl-DL-carnitine chloride (PC). PC was strategically selected for its role as a substrate of the organic cation transporter 2 (OCTN2), enabling active transport across BBB. This approach aimed to enhance the physicochemical stability, permeability, and GBM selectivity of the A1874-loaded PROnano formulation. By facilitating efficient translocation into the brain and accumulation at the tumor site, this target strategy addresses major limitations in GBM therapy and supports the development of precision-guided nanoplatform for PROTAC-based interventions.

Methods: Extensive pre-formulation studies were conducted to determine the solubility profile of A1874 in various pharmaceutically acceptable media. Aqueous buffers with pH values of 1.2, 3.0, 5.5, 6.8, and 7.4 were evaluated to assess pH-dependent solubility behavior. Additionally, biocompatible organic solvents including dimethylacetamide(DMA), polyethylene glycol 400(PEG 400), propylene glycol(PG), and benzyl alcohol(BA) were screened. Based on solubility, miscibility, and emulsification behavior, DMA, Medium chain triglyceride(Captex 300 MCT), and Kolliphor®ELP were selected for formulation development. To formulate PROnano, drug was initially dissolved in DMA to obtain clear drug solution. This was added to pre-mixed blend of Captex300 MCT and Kolliphor®ELP under vortex agitation to form uniform isotropic preconcentrate. Upon dispersion with deionized water with PC/targeted(PROnano) and without PC/non targeted(Nano), nanoemulsion were spontaneously formed. The dynamic light scattering (DLS) particle size, poly dispersibility index(PDI) and zeta (ζ) potential were analyzed by Malvern zeta sizer. The cytotoxicity of A1874 in DMSO, Nano and PROnano formulation was evaluated in GBM cells and TMZ resistant GBM cells, including U87-MG and U-87 MG/TR (TMZ resistant cells), using the MTT assay. In these cells, the anti-cancer activity of individual inhibitors was also analyzed. To further validate it, quantitative cellular uptake was quantified by reverse-phase high-performance liquid chromatography(RP-HPLC) analysis, while qualitative intracellular distribution was visualized using fluorescence microscopy. Three-dimensional(3D) spheroid models of GBM were employed to evaluate the long-term cytotoxic efficacy of the formulations. Ongoing studies include migration assay and western blot analysis.

Results: A1874 exhibited negligible saturation solubility across all aqueous buffers, including pH 1.2, 3.0, 5.5, 6.8, and 7.4, confirming the need for specialized delivery system. The Nano exhibited particle size of 48.42 nm/0.134, and ζ-potential of –3.5 mV. PRONano showed particle size of 49.54 nm/0.205 and markedly increased positive ζ-potential of +45.5 mV, reflecting successful surface modification(Figure1). Both formulations maintained their physical stability over 24 hours. Cytotoxicity assays conducted in U-87MG, and U-87MG/TR cell lines revealed that A1874, Nano and PRONano has potent anticancer activity, with IC₅₀ values below 600 nM(Figure2). Quantitative intracellular drug accumulation for these groups was assessed using RP-HPLC in U-87 MG cells wherein PROnano achieved significant intracellular accumulation as compared to Nano. Bend.3 cells treated with PROnano exhibited markedly enhanced fluorescence intensity, with pronounced cytoplasmic distribution of the signal, indicating effective internalization across BBB and intracellular trafficking. In contrast, Nano demonstrated considerably weaker fluorescence(Figure2). In 3D spheroid assay of U-87 MG cells, PROnano treatment resulted in substantial decrease in spheroid area, demonstrating strong cytotoxic activity. The non-targeted Nano formulation led to minimal change similar to A1874 DMSO, suggesting marginal efficacy. Live/dead fluorescence imaging performed on Day 8 revealed extensive cell death in the PROnano group, supporting its enhanced therapeutic potential in physiologically relevant tumor model(Figure3).

Conclusion: Collectively, these findings strongly support the feasibility of our proposed approach, demonstrating PROnano as an innovative targeted delivery platform. This innovative platform not only addresses key challenges in GBM therapy, including poor drug solubility and BBB penetration, but also offers a novel approach to improving PROTAC-based treatment efficacy. PROnano holds significant potential for clinical translation, paving the way for improved outcomes in GBM therapy.


Figure 1. (a) Graphical representation of preparation of PRONano (b) Particle size and zeta potential of Nano and PRONano (c) Solubility of A1874 in different biocompatible solvents


Figure 2. Plot of cellular viability of Idasanutlin and JQ1 in (a) U-87 MG cells (b) U-87 MG TR (c) Plot of cellular viability of A1874 (d) Cellular viability of A1874, PRONano and Nano (e) Qualitative cellular uptake of Nano and PRONano in Bend.3 cells confirming its ability to cross BBB (f) Quantitative cellular uptake of A1874 in DMSO, PRONano and Nano in GBM cells U-87 MG confirming nanoformulation uptake at tumor site


Figure 3. In vitro efficacy in 3D spheroid model of U-87 MG cells (a) Representative images of 3D multicellular spheroids of U-87 MG cells following 8 days of treatment (b) Change in area of spheroids with respect to time in the control, A1874 DMSO, Nano and PRONano group at 200 nM concentration (c) Apoptosis assay of 3D multicellular tumor for control, A1874 DMSO, Nano and PRONano. Scale bar = 530 μm. (n = 3)